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Desrisking the Cytotoxicity of a Mitochondriotropic Antioxidant Based on Caffeic Acid by a PEGylated Strategy Carlos Fernandes, Sofia Benfeito, Ricardo Amorim, Jose Teixeira, Paulo J Oliveira, Fernando Remião, and Fernanda Borges Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00383 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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Bioconjugate Chemistry

Desrisking the Cytotoxicity of a Mitochondriotropic Antioxidant Based on Caffeic Acid by a PEGylated Strategy

Carlos Fernandes†, Sofia Benfeito†, Ricardo Amorim†‡, José Teixeira†‡, Paulo J. Oliveira‡, Fernando Remião§, Fernanda Borges†*

†

CIQUP-Department of Chemistry and Biochemistry, Faculty of Sciences, University

of Porto, 4169-007, Porto, Portugal. ‡

CNC-Center for Neuroscience and Cell Biology, UC-Biotech, University of Coimbra,

Biocant Park, Cantanhede 3060-197, Portugal §

UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences,

Faculty of Pharmacy, University of Porto, 4050-313, Porto, Portugal.

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Mitochondrial oxidative damage is related to diverse pathologies, including cancer and neurodegenerative diseases. Shielding mitochondria from oxidative damage with mitochondriotropic antioxidants is by now considered an effective therapeutic strategy. Despite the success of the approach some concerns related with cytotoxicity have been reported. For instance, AntiOxCIN6, is a mitochondriotropic antioxidant based on caffeic acid (CAF) that is cytotoxic in hepatocarcinoma (HepG2) cell lines. PEGylation, often used to enhance drug pharmacologic and pharmaceutical properties, was herein applied to modulate AntiOxCIN6 toxicity drawbacks. So, a dualfunctionalization of polyethylene glycol (PEG) with TPP+ and CAF as targeting and antioxidant arms, respectively, was performed by a two-step amidation strategy using ethyl chloroformate and EDC/NHS as coupling reagents. The data showed that the antioxidant properties related with CAF moiety were maintained in the CAF-PEG-TPP conjugate (CPTPP) and that PEGylation process reverted the loss of ability to chelate iron observed with AntiOxCIN6.. In cellular studies, CPTPP was non-toxic to human HepG2 and neuronal (SH-SY5Y) cells, while both CAF and AntiOxCIN6 demonstrated harmful effects in the same cell lines. The lack of cytotoxic events linked to oxidative stress levels observed with CPTPP suggested that PEGylation process somehow modulate the putative toxicity related with the presence of a catechol moiety and/or the TPP+ cation. In addition, the mitochondrial oxygen consumption was not significantly affected by CPTPP-treatment in SH-SY5Y cells when compared with non-treated cells. CPTPP showed remarkable antioxidant effects in cell-based assays against several oxidative stress-induced agents (H2O2, t-BHP and FeNTA). From the data it can be concluded that PEGylation technology can modulate the toxicity of mitochondriotropic antioxidants without disturbing the antioxidant profile of the core antioxidant. PEGylation can be considered a relevant tool to hasten the difficulties related to the design and development of mitochondrial nontoxic and operative drug candidates.


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INTRODUCTION Mitochondria are the powerhouses of cells that sustain the cellular homeostasis providing cellular energy, stored in adenosine triphosphate (ATP), via oxidative phosphorylation (OXPHOS).1 During mitochondrial oxidative metabolism reactive oxygen species (ROS) are generated by endogenous redox (reduction/oxidation) processes.2 When ROS are over-expressed without up-regulated antioxidant protection mitochondrial dysfunction occurs, a shortcoming linked with a variety of oxidativestress related disorders, such as neurodegenerative diseases, ischemia−reperfusion damage and cancer.3 Regardless of the clinical significance of these diseases, many of them still lack therapeutic solutions.4 One therapeutic strategy proposed to reduce or prevent the mitochondrial oxidative damage is related to the targeting of antioxidants to mitochondria. Mitochondriotropic antioxidants are generally based on the conjugation of a bioactive compound to a delocalized triphenylphosphonium (TPP+) cation.5,6 Through this approach, a 5−10-fold higher accumulation of the active molecule is normally measured in the cytoplasm, with an additional hundred or thousand-fold accumulation in mitochondria.5-7 Recently, natural hydroxycinnamic acids (HCAs) present in diet were functionalized with a TPP+ cation, a carrier that successfully drive their accumulation inside mitochondria.8 They can work as multi-functional antioxidants as the core HCAs are described to be operate by a combination of mechanisms, namely (a) direct of dietary antioxidants scavenging of ROS, (b) chelation of transition metals (e.g. Cu and Fe), (c) induction of cytoprotective signalling pathways.9 Despite the successful approach based on directing antioxidants to mitochondria by using TPP+ moiety, some concerns related with their cytotoxicity have been reported. 6, 10-12

The effects were related to mitochondria disruption mostly caused by the

accumulation of the lipophilic cation. For instance, Cortes et al. reported that gallate TPP+-based compounds at 1 µM caused a significant change of the mitochondrial transmembrane, indicating the existence of a transitory phenomenon, such as the mitochondrial permeability transition pore.11Teixeira et al. found that the conjugation of an alkyl TPP+ chain with caffeic acid (CAF), a well-known HCA, resulted in a compound (AntiOxCIN6) with significant cytotoxic effects in hepatocarcinoma (HepG2) cell lines.8 PEGylation is a classically defined as a strategy based on the covalent attachment to a protein, peptide or non-peptide chemical entity to one, or more, polyethylene glycol 3 ACS Paragon Plus Environment


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(PEG) chains. PEG is a polymer highly soluble in water, approved by Food and Drug Administration, known to be nontoxic, non-immunogenic and non-antigenic.13 The PEG-drug conjugation has been reported as an effective therapeutic solution to increase the efficacy of drugs in clinical settings, namely by modulating the molecular size and solubility of a drug and decrease drug degradation by metabolic enzymes, among others. Based on these assets several PEGylated drugs are already being used in the clinic.14 Although PEGylation is often used to enhance pharmacologic and pharmaceutical properties, few research has been performed toward its benefice on the modulation of toxicity shortcomings. In this context, our group have reported the beneficial effect of PEGylation to decrease CAF cytotoxicity outcomes while maintaining its in vitro neuroprotective properties.15 Taken in consideration the importance of mitochondrial antioxidant strategy and PEGylation paybacks it is thought that this technology can also address the problems reported in targeting drugs to mitochondria, like drug payloads, pharmacokinetics and toxicity drawbacks. In line, the effect of PEGylation to circumvent the toxicity of the mitochondriotropic antioxidant based on caffeic acid AntiOxCIN6, was herein studied. Specifically, the alkyl spacer between TPP+ and CAF was replaced by a PEGylated moiety using a carbodiimide crosslinker strategy. The cytotoxic profile and antioxidant properties in non-cell and cellular systems of the new conjugate system, named CAFPEG-TPP+ (CPTPP), were evaluated and compared with AntiOxCIN6, and CAF.

RESULTS AND DISCUSSION Chemistry. The route followed to synthesize CPTPP is described in Scheme 1 (route a and b). First, compound (1) was obtained

by amidation of tert-butyl (5-

aminopentyl)carbamate with (5-carboxypentyl)triphenylphosphonium, in a process that encloses carboxylic acid activation by POCl3 and DIPEA under mild conditions (Scheme 1, route a).16 Then, CAF was activated with ethyl chloroformate in the presence of triethylamine in order to perform the amidation reaction with H2N-PEGCOOH (Scheme 1, route b).17 Subsequently the PEG carboxyl group was activated with EDC and NHS and coupled by an amidation reaction with compound (1).


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Scheme 1. Strategy followed to synthesize (6-((5-aminopentyl)-amino)-6-oxohexyl)triphenylphosphonium (a) and CPTPP (b)a

a

Reagents and conditions: i) DIPEA, POCl3, N-Boc-1,6-hexanediamine hydrochloride, r.t.,

overnight; ii) concentrated HBr, rt, 24 h; iii) triethylamine, ethyl chloroformate, H2N-PEGCOOH (Mw ~ 3.5 kDa), rt, 24 h; iv) compound (1), EDC, NHS, rt, 48 h.

In that case a complete activation reaction of PEG end group was crucial to avoid the presence of non-functionalized PEG or polymer cross-linking side reactions.18 So, the activation of PEG-TPP moiety was performed using an excess of coupling reagent. The reagents in excess were removed by dialysis. The synthesis of AntiOxCIN6 was performed by a four step synthetic strategy as previously described.8 Structural characterization of CPTPP. CPTPP structural characterization was performed by proton nuclear magnetic resonance (1H NMR) and matrix assisted laser 5 ACS Paragon Plus Environment


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desorption ionization-time-of-flight (MALDI/TOF) mass spectrometry. Both methods have been shown to be useful in the characterization of 15

PEGylated phenolic

1

conjugates. CPTPP and compound (1) H NMR spectra are shown in Figure 1a.

Figure 1. Characterization CPTPP data (a) 1H NMR spectra of compound (1) and CPTPP and respective magnification between 6 and 9 ppm (spectra inset); (b) MALDI-TOF spectra of CPTPP and H2N-PEG-COOH). Polytools Software was used to calculate the average number molecular weight (Mn) values.

To compare the 1H NMR data, the spectra of compounds (1) and CPTPP were normalized (Figure 1a). However, due to the lengthy of PEG chain of CPTPP conjugate all peaks resultant from CAF and/or TPP+ cation, as they have great Mw differences, were difficult to notice.19 Thus, as shown in Figure 1a, compound (1) spectrum showed signals between 1.1 and 3.3 ppm attributed to the alkyl chain (22H) located between TPP+ and amine moiety, aromatic ring peaks related with TPP+ moiety (15H) and the peak of amide bond (1H) between 7.7 and 8.0 ppm.8 The signals of alkyl chain and TPP+ also appeared in NMR spectra of CPTPP conjugate, as well as the signal of PEG backbone at 3.6 ppm (Figure 1a).20 In addition, signals positioned at 7.2 and 6.8 ppm due to the phenyl protons (3H) as well as the peaks of Hα (6.2 ppm, 1H, J=16 Hz) and Hβ (7.5 ppm, 1H, J=16 Hz) from double bond were observed. Comparing the intensity of the peaks related with Hα and with the protons from the aromatic rings of TPP moiety, a ratio of 1:15 of (IHα:HTFF) was estimated, which is in accordance with the expected. 6 ACS Paragon Plus Environment

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The Mn of CPTPP was measured using MALDI/TOF (Figure 1b). The MALDI-TOF spectra clearly showed an increase of Mn values after functionalization of H2N-PEGCOOH (3511.5 Da) with CAF (180.2 Da) and TPP+ (471.6 Da) moieties, having CPTPP conjugate a Mn value of 4223.3 Da. As observed, the CPTPP Mn value obtained by MALDI-TOF and the predicted values using the Mn of H2N-PEG-COOH and the mass of increments are slight different.21,22 This could be due to the formation of adducts between PEG and sodium [M + Na]+ or potassium [M + K]+ in CPTPP, since PEG has high affinity with alkali salts.15 The combination of data from NMR and MALDI-TOF analysis confirm the successful functionalization of PEG with both phenolic antioxidant (CAF) and TPP+ moieties. CPTPP radical scavenging properties. Phenolic compounds have been widely reported as antioxidants, being this property attributed to diverse mechanisms.23,24 To verify the antioxidant activity of CPTPP its radical scavenging activity was evaluated towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS●+) and 2,2diphenyl-1-picrylhydrazyl (DPPH● ) radicals using Trolox as reference compound (Figure 2a). The concentration of CPTPP was determined taking into account the value of Mn obtained by MALDI-TOF analysis. The same experiences were performed with CAF and AntiOxCIN6.

**

****

Figure 2. Antioxidant activity of CPTPP, CAF and AntiOxCIN6 towards ABTS●+ and DPPH● radicals (a) and iron (II) (b). Trolox and ethylenediamine tetraacetic acid (EDTA) were used as reference compounds in ABTS●+/DPPH● and iron chelation assays, respectively. Data are means ± SD of three independent experiments and the results are expressed as IC50 (µM) or percentage



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(%) of iron (II) chelation values for ABTS●+/DPPH● and iron chelation assays, respectively. In all cases, p values lower than 0.05 were considered significant (**p

Desrisking the Cytotoxicity of a Mitochondriotropic ... - ACS Publications

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